A Sigmoidal Model for the Interpretation of Quantitative PCR (QPCR) Experiments

• Gutiérrez Sánchez, Pablo Andrés ^[1] ; Rodríguez Fuerte, Verónica ^[1] ; Marín Montoya, Mauricio ^[1]
  1. [1] Universidad Nacional de Colombia

     Universidad Nacional de Colombia

     Colombia

     
• Localización: Revista de la Facultad de Ciencias Básicas, ISSN-e 1900-4699, Vol. 8, Num. 2, 2012, págs. 244-253
• Idioma: inglés
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• Resumen

  • Real-time or quantitative PCR (qPCR) is the most commonly used technique for estimating the amount of starting nucleic acids in a PCR or RT-PCR reaction. Quantification of PCR product is achieved in real time by measuring the increase in fluorescence of intercalating dyes, labeled primers or oligonucleotides in the presence of double stranded DNA. This amplification curve follows a sigmoid behavior and is used to estimate the relative and/or absolute amount of template using different methods and assumptions. Estimation of C0 normally requires the measurement of a threshold cycle and some assumption about the efficiency of the reaction. An accurate estimation of efficiency is paramount for a precise determination of template levels at time zero. Several non-linear fitting methods have been implemented to model the sigmoid behavior using different empirical models with varying amounts of parameters; however, interpretation of the corresponding parameters is not straightforward. In this paper a model of PCR amplification is deduced and used in the interpretation of qPCR experiments. A non-linear regression analysis of this equation gives a direct estimation of C0 and automatically calculates a parameter k related to the reaction efficiency. This model takes into account non-idealities in the amplification reaction and avoids a priori assumptions about efficiency.